CN113293121B - Intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli - Google Patents
Intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli Download PDFInfo
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Abstract
The invention provides an intelligent regulation and control method for carbon metabolic flux of xylitol produced by escherichia coli, aiming at realizing high-efficiency synthesis of xylitol in the process of preparing xylitol by utilizing a cell factory. The SecY engineered escherichia coli is successfully constructed by introducing a SecY non-specific sugar transport channel and simultaneously overexpressing heterologous xylose reductase (CbXR). The strain can overcome CCR effect through self-regulation, and relieve the inhibition effect of the xylitol 5-phosphate on the self-transported xylose. Changes of external carbon sources are used as response signals, the CCR effect and the inhibition effect of the xylitol 5-phosphate are converted into two important regulation switches in a carbon metabolism network, and the metabolic capacity of cells on glucose and xylose is regulated and controlled. The method can completely metabolize glucose and xylose in any proportion in the substrate, ensures the maximum conversion efficiency of energy to the target product, and better meets the requirement of green biological manufacturing.
Description
Technical Field
The invention belongs to the field of microbial metabolic engineering, and particularly relates to an intelligent carbon metabolic flux control method for producing xylitol by using escherichia coli.
Background
Biorefineries are beneficial for the protection of the environment and the protection of non-renewable fossil fuels, and industrial biotechnology can convert carbon sources in agricultural wastes into valuable chemicals, materials, and biofuels to achieve sustainable economic development. Xylitol is a five-carbon sugar alcohol, as sweet as sucrose, and occurs naturally in fruits, vegetables, and mushrooms. Xylitol is considered a valuable derivative in the food, pharmaceutical and chemical industries because of its low calorie, tooth-protecting, anti-diabetic and anti-carcinogenic properties. The chemical production of xylitol mainly uses xylan as raw material to make artificial production. On the contrary, the production of xylitol by microbial fermentation is more environment-friendly and has lower cost.
However, two important factors cannot be avoided in the production of xylitol in a mixed carbon source of glucose and xylose by using E.coli by means of metabolic engineering techniques. Firstly, carbon catabolism repression effect (CCR effect for short) generated by the existence of glucose inhibits the expression of xylose transporter, so that xylose cannot be smoothly transported into cells; secondly, xylitol is also phosphorylated by xylulokinase to produce xylitol 5-phosphate, which exerts feedback inhibition on xylose transporters. This is also the reason for the low xylitol production.
Although the existing method respectively overcomes CCR effect and xylitol-5-phosphate inhibition in escherichia coli genetic engineering strains to realize the transformation of xylitol, enough glucose needs to be additionally added to escherichia coli to ensure the continuous demand of cells on reducing power. However, the original biomass is reused after pretreatment, the carbon source proportion is random, and how to rationally design the carbon metabolic pathway of escherichia coli, so that the conversion of energy to xylitol is maximized is a key problem to be solved.
Disclosure of Invention
The invention aims to solve the problems and provides an intelligent regulation and control method for carbon metabolic flux of xylitol produced by escherichia coli. The engineering escherichia coli is constructed by constructing an escherichia coli genetic engineering strain SecY, the CCR effect and the inhibition effect of 5-phosphate xylitol in the process of producing xylitol by fermenting escherichia coli are overcome, the distribution of carbon metabolic flux is rationally designed, and the yield of producing xylitol by a microbial fermentation method is improved.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
SecY non-specific sugar transport channels are successfully constructed in Escherichia coli JM109 (DE 3), so that xylose is ensured to freely diffuse into cells under the action of concentration difference, a metabolic pathway from xylose to xylitol is constructed, and the efficient fermentation production of xylitol by utilizing a mixed carbon source by Escherichia coli is realized.
Over-expressionsecY(ΔP)、secE、secGAndsCVEfour genes in knockoutxylFGenes andxylEescherichia coli JM109 (DE 3) (i.e., escherichia coli JM109 (DE 3). DELTA. XylF-. DELTA.XylE) of the gene) succeeded in constructing a SecY nonspecific sugar transport channel. Over-expression on the basis of the abovecbXRThe SecY engineered escherichia coli is successfully constructed by the gene, the metabolic pathway of reducing xylose into xylitol is realized in the escherichia coli, and the inhibition of the xylitol-5-phosphate on cell metabolism is effectively relieved. The use of different ratios of glucose and xylose for fermentation illustrates that SecY is engineered to be largeEnergy metabolism mode of enterobacter in intelligent regulation and control of carbon metabolism flux.
An intelligent regulation and control method for carbon metabolism flux of xylitol produced by escherichia coli comprises the following steps:
(1) Overexpression of genes in JM109 (DE 3) Δ XylFGH- Δ XylE E.colisecY(ΔP)、sCVE、secEAndsecGconstructing and expressing a SecY non-specific sugar transport channel;
(2) Overexpression in E.coli containing SecY non-specific sugar transport channelscbXRGene, synthetic route for constructing and expressing xylitol;
(3) Glucose and xylose in different proportions are used as main conditions for fermentation, and the effect of the intelligent carbon metabolic flux regulation and control system is clarified.
The JM109 (DE 3) Δ XylF- Δ XylE E.coli in the above step (1) is constructed by a method comprising the steps of: are respectively designed to containxylFGene, gene,xylEPCR primers of the gene homologous arm sequence are subjected to PCR amplification by taking a pkD13 plasmid as a template to obtain a KANA fragment with two ends containing a homologous arm and an FRT recognition site, and then transfected into a JM109 (DE 3) competent cell which contains a pKD46 plasmid and is induced by L-arabinose; verification of successful replacement of KANA fragment by using genome sequence design PCR primerxylFGenes andxylEthe gene was then transfected with pCP20 plasmid for specific recognition of FRT site, eliminating KANA gene, and obtaining JM109 (DE 3). DELTA.XylF-. DELTA.XylE E.coli.
In the above step (1), the genesecY(ΔP)、sCVE、secEAndsecGthe nucleotide sequences of (A) are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Constructing and expressing a SecY non-specific sugar transport channel in the step (1), wherein the specific process comprises the following steps: four groups of primers with corresponding restriction enzyme sites are used for respectively pairingsecY(ΔP)、secE、secGAndsCVEafter four genes are subjected to PCR amplification to obtain four gene fragments, the four genes are subjected to PCR amplificationsecY(Δ p) andsecEthe gene was ligated with pETDuet plasmid,secGandsCVEthe gene was ligated with prsfDuet plasmid to obtain recombinant vector pETDuet-secY(Δp)-secEAnd prsfDuet-secG-sCVE(ii) a Recombinant vector pETDuet-secY(Δp)-secEAnd prsfDuet-secG-sCVEThe SecY non-specific sugar transport channel is expressed in Escherichia coli genetic engineering bacteria after being transfected into JM109 (DE 3) delta XylF-delta XylE Escherichia coli cells.
The synthesis route for constructing and expressing the xylitol in the step (2) comprises the following specific processes: will be provided withcbXRGene ligation to recombinant vector prsfDuet-secG-sCVEThe successful construction of the recombinant vector prsfDuet-secG-sCVE-cbxR(ii) a Transfection of recombinant vector pETDuet-secY(Δp)-secEAnd prsfDuet-secG-sCVE-cbxR(ii) a Successful overexpression in E.coli cellscbXRAfter exogenous gene, secY engineered Escherichia coli is obtained, namely JM109 (DE 3) delta XylF-delta XylE carries pETDuet-secY(Δp)-secEAnd prsfDuet-secG-sCVE-cbxR(ii) a The above-mentionedcbXRThe nucleotide sequence of the gene is shown in SEQ ID NO. 5.
In the step (3), the fermentation medium is prepared by setting glucose and xylose with different proportions in an M9 medium, and the fermentation conditions are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 60 hours, the rotating speed of a shaking table is 220rpm, and the concentration of an inducer IPTG is 0.4mM. Preferably, the concentrations of glucose and xylose are: 0 mM and 100 mM, or 33 mM and 66 mM; or 50 mM and 50 mM.
Further, referring to fig. 1, the "intelligent regulation and control of carbon metabolic flux" in step (3) can be self-regulated after recognition of sugar signals by the engineered escherichia coli. In the first stage (A), a phosphoenolpyruvate-sugar phosphotransferase system (PTS system for short) strictly controls the expression of xylose-related genes while transporting glucose, and xylose enters cells through a SecY nonspecific sugar transport channel to start producing xylitol; in the second phase (B), only xylose remains in the medium, activating the genes involved in xylose metabolism, and the produced xylitol-5-phosphate immediately blocks the transport function of xylose. After xylose crosses the plasma membrane, one portion enters the pentose phosphate pathway to power the cell, while the other portion continues to support the production of xylitol.
The carbon metabolism flux intelligent control method for producing xylitol by using escherichia coli is applied to the production of xylitol.
Compared with the prior art, the invention has the following beneficial effects:
in the present invention, xylose transport does not need to rely on native xylose transporters, and the inhibition of CCR effect and xylitol-5-phosphate can be relieved only by SecY non-specific sugar transport channels. Meanwhile, the complete utilization of raw materials is realized to the maximum extent, glucose and xylose in any proportion can continuously enter the metabolic flow of SecY engineered escherichia coli under the action of intelligent regulation and control of carbon metabolic flow, and the production of xylitol is not limited by the content of glucose any more.
Drawings
FIG. 1 is a flow chart of intelligent regulation of carbon metabolic flux.
FIG. 2 is a schematic view of a display devicexylFAndxylEschematic diagram of the knockout result of (c). In A, left: 1 is a standard strip; 2-5 are KANA fragments containing homology arms; the method comprises the following steps: 1 is a standard strip; 2-5 for verifying genomexylEThe gene position was successfully replaced by a KANA fragment; and (3) right: successfully transfecting the pCP20 plasmid, identifying an FRT site, and knocking out a KANA1 fragment on a genome; 1 is a standard strip; 2-5 are verification of anti-disease fragments. In B, left: 1 is a standard strip; 2-5 is a KANA fragment containing homology arms; the method comprises the following steps: 1 is a standard strip; 2-9 for verifying genomexylFThe gene position was successfully replaced by the KANA fragment; and (3) right: successfully transfecting the pCP20 plasmid, identifying an FRT site, and knocking out a KANA1 fragment on a genome; 1 is a standard strip; 2-9 are verification of anti-disease fragments.
FIG. 3 SecY non-specific sugar transport channels efficiently transport xylose. A, cell growth curve; b-consumption of xylose in the medium.
FIG. 4 SecY engineered E.coli for the efficient synthesis of xylitol. Xylose consumption curve; xylitol is a Xylitol generation curve; OD 600 : cell growth curves.
FIG. 5 shows the sugar metabolism and xylitol synthesis ability under the intelligent control of carbon metabolism flux. Glucose consumption curve; xylose consumption curve; xylitol is a Xylitol generation curve; OD 600 : cell growth curves.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting.
Example 1
Knockdown in Escherichia coli JM109 (DE 3)xylFAndxylE，completely cutting off the xylose transferring capacity in the strain body and limiting the strain to metabolize xylose. Knocking out escherichia coli natural xylose transporter by using lambda red homologous recombination technologyxylFAndxylEthe purpose is to verify the function of SecY non-specific sugar transport channels in genetic engineering strains.
The specific knockout process comprises the following steps:
PCR primers containing homologous arm sequences are respectively designed, pkD13 plasmid is used as a template for PCR amplification, a KANA fragment containing the homologous arm and an FRT recognition site at two ends is obtained, and then the KANA fragment is transfected into JM109 (DE 3) competent cells which contain pKD46 plasmid and are induced by L-arabinose and cultured at 37 ℃. Utilizes the genome sequence to design a PCR primer to verify that the KANA fragment (the nucleotide sequence of which is shown as SEQ ID NO. 6) is successfully replacedxylFGenes andxylEthe gene was then transfected into pCP20 plasmid for specific recognition of FRT site and elimination of KANA gene to obtain JM109 (DE 3). DELTA.XylF-. DELTA.XylE E. The results in figure 2 show that,xylFandxylEthe knockout was successful.
The PCR primers involved are as follows:
example 2 construction of construction and expression of SecY non-specific sugar transport channels
Selecting JM109 (DE 3) delta XylF-delta XylE escherichia coli as a chassis host, and co-expressing plasmids pETDuet-1 and pRSFDuet-1 through a recombinant vectorsecY(ΔP)、secE、secGAndsCVEfour genes.
As described abovesecY(ΔP)Obtaining the gene, which comprises the following steps: genetic engineering by means of overlapping PCRConstructing the K family strain of Escherichia colisecYMutant gene after 60-74 site amino acid in gene is replaced by flexible segment containing four amino acids of GlySerGlySersecY(ΔP)，secY(ΔP)The nucleotide sequence of the gene is shown in SEQ ID NO.1。 secYGene NCBI Gene ID:947799. the primers involved in the overlap PCR were as follows:
as described abovesCVEObtaining the gene, which comprises the following steps: according to the preference of colibacillus coding amino acid, after codon optimization, artificially synthesizingsCVEThe nucleotide sequence of the gene is shown in SEQ ID NO. 2.
The secE gene described aboveAndsecGthe nucleotide sequences of the genes are shown in SEQ ID NO.3 and SEQ ID NO.4, and the NCBI Gene IDs are 948486 and 947720 respectively.
The specific construction process is as follows: four groups of primers with corresponding restriction enzyme sites are used for respectively pairingsecY(ΔP)、secE、secGAndsCVEcarrying out PCR experiment on the four genes to obtain fragments of the four genes, carrying out double enzyme digestion on the plasmid and the gene fragments respectively, recovering the fragments, connecting the fragments overnight at 16 ℃ by using T4DNA ligase, and obtaining a recombinant vector pETDuet-secY(Δp)-secE；prsfDuet-secG-sCVE. The PCR primers involved are as follows:
transfection of recombinant vector pETDuet-secY(Δp)-secEAnd prsfDuet-secG-sCVEAnd introducing a SecY non-specific sugar transport channel for fermentation to realize the expression of the SecY non-specific sugar transport channel in the escherichia coli genetic engineering bacteria. The fermentation medium is a medium prepared by adding 4g/L xylose into M9, the strain LB test tube is activated overnight, the inoculation concentration OD600=0.1, the culture is carried out at 30 ℃, the culture is carried out at 220rpm, and the concentration of an inducer IPTG is 0.4mM. Fermenting for 96 hours in 250ml shake flaskSamples were taken once every 12 hours.
The formula of the M9 culture medium is as follows: 7.52 g/L Na 2 HPO 4 ·2H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 0.5 g/L NH 4 Cl, 0.25 g/L MgSO 4 ·7H2O, 44.1 mg/L CaCl 2 ·2H 2 O, 1 mg/L biotin, 1 mg/L thiamin, 50 mg/L EDTA, 8.3 mg/L FeCl 3 ·6H 2 O, 0.84 mg/L ZnCl 2 , 0.13 mg/L CuCl 2 ·2H 2 O, 0.1 mg/L CoCl 2 ·2H 2 O, 0.1 mg/L H 3 BO 3 And 0.016 mg/L MnCl 2 ·4H 2 O。
The results in FIG. 3 show that in the strains without SecY non-specific sugar transport channels, the cells were arrested in growth and xylose was not metabolized significantly; and a SecY non-specific sugar transport channel is introduced, so that the xylose transport of the cells is guaranteed, and the cells recover normal growth. The experimental result shows that the SecY non-specific sugar transport channel has xylose transport capacity.
Example 3 construction and expression of synthetic pathway for xylitol overexpressioncbXRGene
cbXRThe gene is fromCandida boidiniiThe gene is artificially synthesized after codon optimization and optimizedcbXRThe nucleotide sequence of the gene is shown as SEQ ID NO. 5. Will be provided withcbXRGene ligation to recombinant vector prsfDuet-secG-sCVEThe successful construction of the recombinant vector prsfDuet-secG-sCVE-cbxR。
Transfection of recombinant vector pETDuet-secY(Δp)-secEAnd prsfDuet-secG-sCVE-cbxR。Successful overexpression in E.coli cellscbXRAfter exogenous gene, secY engineered Escherichia coli is obtained, namely JM109 (DE 3) delta XylF-delta XylE carries pETDuet-secY(Δp)-secEAnd prsfDuet-secG-sCVE-cbxR. The xylitol synthesis is preliminarily verified in an LB medium (5 g/L yeast powder, 10 g/L peptone, 10 g/L NaCl). 50 mM xylose was added to the medium at 30 ℃ and a concentration of 0.4mM IPTG, a culture inducer at 220 rpm. The 250ml shake flask was fermented for 72 hours, and a sample was taken once for 12 hours. The experimental results are as followsFIG. 4, 50 mM xylose was completely metabolized and 19.5 mM xylitol was produced. The production of xylitol in large quantities also indicates that the transport of xylose by the cells is not limited by xylitol-5-phosphate. SecY non-specific sugar transport channels have a positive promoting effect on the synthesis of xylitol.
Example 4
SecY engineering colibacillus is fermented in an M9 culture medium, and three groups of glucose and xylose are set in different proportions: 0 mM and 100 mM;33 mM and 66 mM;50 mM and 50 mM. The inducer IPTG was cultured at 220rpm at 30 ℃ at a concentration of 0.4mM. The 250ml shake flask was fermented for 72 hours and a sample was taken once at 12 hours. The analysis of experimental results shows that in the first stage of intelligent regulation and control of carbon metabolic flux, the yield of xylitol in a phosphoenolpyruvate-sugar phosphotransferase system (PTS system for short) is 70.3 percent; in the second stage of intelligent regulation and control of carbon metabolism flow, the yield of xylitol is 27.9%. In the second stage of intelligent regulation, 72.1% of xylose enters glycolysis pathway to supply energy for cell growth and xylitol synthesis. FIG. 5 shows the metabolism of glucose and xylose at 33 mM and 66 mM, respectively, from which it can be seen that glucose and xylose begin to be metabolized at almost the same time, indicating that the CCR effect has been abolished by SecY non-specific sugar transport channels, with a final xylitol production of 30 mM. Glucose and xylose are almost completely metabolized within 60 hours, and the advantageous effects of the intelligent carbon metabolic flux regulation system are further explained.
The above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fuzhou university
<120> carbon metabolic flux intelligent control method for xylitol production by escherichia coli
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gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact 540
ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc 600
agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 660
gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg 720
tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca 780
gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc 840
ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 900
tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt 960
actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt 1020
gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc 1080
tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg 1140
ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg 1200
cgccctgagt gcttgcggca gcgtgagctt caaaagcgct ctgaagttcc tatactttct 1260
agagaatagg aacttcgaac tgcaggtcga cggatccccg gaat 1304
<210> 7
<211> 71
<212> DNA
<213> Artificial sequence
<400> 7
acagctcgct ctctttgtgg aatccgtctt taattaccgt atctttgatg gtgtaggctg 60
gagctgcttc g 71
<210> 8
<211> 70
<212> DNA
<213> Artificial sequence
<400> 8
ccatgaaaat aaagaacatt ctactcaccc tttgcacctc actcctgctt attccgggga 60
tccgtcgacc 70
<210> 9
<211> 71
<212> DNA
<213> Artificial sequence
<400> 9
taccttagtc gctacattag gtggtttatt atttggctac gacaccgccg gtgtaggctg 60
gagctgcttc g 71
<210> 10
<211> 70
<212> DNA
<213> Artificial sequence
<400> 10
ttacagcgta gcagtttgtt gtgttttctt cgtttccggt tcccagagcg attccgggga 60
tccgtcgacc 70
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence
<400> 11
cagcaagcga accggaattg ccagc 25
<210> 12
<211> 27
<212> DNA
<213> Artificial sequence
<400> 12
gccaattcat tcacgcggca tggagag 27
<210> 13
<211> 25
<212> DNA
<213> Artificial sequence
<400> 13
cagcaagcga accggaattg ccagc 25
<210> 14
<211> 26
<212> DNA
<213> Artificial sequence
<400> 14
cctgtggctg tgtaattcga aacggc 26
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence
<400> 15
atggctaaac aaccgggatt ag 22
<210> 16
<211> 40
<212> DNA
<213> Artificial sequence
<400> 16
agatagaagc gctgcccgaa ccggtgcctc gctgttgctc 40
<210> 17
<211> 42
<212> DNA
<213> Artificial sequence
<400> 17
agcgaggcac cggttcgggc agcgcttcta tctttgctct gg 42
<210> 18
<211> 19
<212> DNA
<213> Artificial sequence
<400> 18
ttatcggccg tagcctttc 19
<210> 19
<211> 30
<212> DNA
<213> Artificial sequence
<400> 19
catgccatgg ctaaacaacc gggattagat 30
<210> 20
<211> 44
<212> DNA
<213> Artificial sequence
<400> 20
cgcggatcct tagtggtgat gatggtgatg tcggccgtag cctt 44
<210> 21
<211> 31
<212> DNA
<213> Artificial sequence
<400> 21
tatacatatg agtgcgaata ccgaagctca a 31
<210> 22
<211> 32
<212> DNA
<213> Artificial sequence
<400> 22
cggggtacct tatcagaacc tcaggccagt ga 32
<210> 23
<211> 38
<212> DNA
<213> Artificial sequence
<400> 23
catgccatgg tgatgtatga agctctttta gtagtttt 38
<210> 24
<211> 33
<212> DNA
<213> Artificial sequence
<400> 24
cgcggatcct tagttcggga tatcgctggt cgg 33
<210> 25
<211> 33
<212> DNA
<213> Artificial sequence
<400> 25
tatacatatg tactcgttcg tatctgaaga aac 33
<210> 26
<211> 32
<212> DNA
<213> Artificial sequence
<400> 26
cggggtacct taaaccagca ggtccgggac ac 32
Claims (7)
1. An intelligent regulation and control method for carbon metabolism flux of xylitol produced by escherichia coli is characterized by comprising the following steps: secY non-specific sugar transport channels are successfully constructed in Escherichia coli JM109 (DE 3), so that xylose is freely diffused into cells under the action of concentration difference, a metabolic pathway from xylose to xylitol is constructed, and the efficient fermentation production of xylitol by utilizing a mixed carbon source through Escherichia coli is realized;
the method comprises the following steps:
(1) Overexpression of genes in JM109 (DE 3) Δ XylF- Δ XylE E.colisecY(ΔP)、sCVE、secEAndsecGconstructing and expressing a SecY nonspecific sugar transport channel; the genesecY(ΔP)、sCVE、secEAndsecGthe nucleotide sequences of (A) are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4;
(2) In a SecY-containing non-specific sugar transport channelIs overexpressed in Escherichia colicbXRGene, synthetic route for constructing and expressing xylitol; the above-mentionedcbXRThe nucleotide sequence of the gene is shown as SEQ ID NO. 5;
(3) Glucose and xylose in different proportions are used as main conditions for fermentation culture, and the effect of the carbon metabolic flux intelligent control system is clarified.
2. The intelligent regulation and control method for carbon metabolic flux of xylitol produced by escherichia coli according to claim 1, wherein the method comprises the following steps: the JM109 (DE 3) Δ XylF- Δ XylE Escherichia coli described in the step (1) is constructed by a method comprising the steps of: are respectively designed to containxylFThe gene,xylEPCR primers of the gene homologous arm sequence are subjected to PCR amplification by taking a pkD13 plasmid as a template to obtain a KANA fragment with two ends containing a homologous arm and an FRT recognition site, and then transfected into a JM109 (DE 3) competent cell which contains a pKD46 plasmid and is induced by L-arabinose; design of PCR primers Using genomic sequences verification of successful replacement of KANA fragmentxylFGenes andxylEthe gene was then transfected into pCP20 plasmid for specific recognition of FRT site and elimination of KANA gene to obtain JM109 (DE 3). DELTA.XylF-. DELTA.XylE E.
3. The intelligent regulation and control method for carbon metabolic flux of xylitol produced by escherichia coli according to claim 1, wherein the method comprises the following steps: constructing and expressing a SecY non-specific sugar transport channel in the step (1), wherein the specific process comprises the following steps: four groups of primers with corresponding enzyme cutting sites are used for respectively pairingsecY(ΔP)、secE、secGAndsCVEafter four genes are subjected to PCR amplification to obtain four gene fragments, the four genes are subjected to PCR amplificationsecY(Δ P) andsecEthe gene was ligated to pETDuet plasmid,secGandsCVEthe gene was ligated with prsfDuet plasmid to obtain recombinant vector pETDuet-secY(ΔP )-secEAnd prsfDuet-secG-sCVE(ii) a Recombinant vector pETDuet-secY(ΔP )-secEAnd prsfDuet-secG-sCVEThe SecY non-specific sugar transport channel is expressed in Escherichia coli genetic engineering bacteria after being transfected into JM109 (DE 3) delta XylF-delta XylE Escherichia coli cells.
4. The intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli as claimed in claim 1, wherein: constructing and expressing a synthetic route of the xylitol in the step (2), wherein the specific process comprises the following steps: will be provided withcbXRGene ligation to recombinant vector prsfDuet-secG-sCVEThe successful construction of the recombinant vector prsfDuet-secG-sCVE-cbxR(ii) a Transfection of recombinant vector pETDuet-secY(ΔP )-secEAnd prsfDuet-secG-sCVE-cbxR(ii) a Successful overexpression in E.coli cellscbXRAfter exogenous gene, secY engineered Escherichia coli is obtained, namely JM109 (DE 3) delta XylF-delta XylE carries pETDuet-secY(ΔP )-secEAnd prsfDuet-secG-sCVE-cbxR。
5. The intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli as claimed in claim 1, wherein: in the step (3), the fermentation medium is prepared by setting glucose and xylose with different proportions in an M9 medium, inoculating SecY engineering escherichia coli for fermentation, and the fermentation conditions are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 60 hours, the rotating speed of a shaking table is 220rpm, and the concentration of an inducer IPTG is 0.4mM.
6. The intelligent regulation and control method for carbon metabolic flux of xylitol produced by escherichia coli according to claim 1, wherein the method comprises the following steps: the concentration of glucose and xylose in step (3) is: 0 mM and 100 mM, or 33 mM and 66 mM; or 50 mM and 50 mM.
7. The use of the intelligent regulation and control method for carbon metabolism flux of xylitol produced by escherichia coli as defined in claim 1 in xylitol production.
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